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| Status |
Public on Jul 14, 2023 |
| Title |
MALBAC_GM12878_rep4 |
| Sample type |
SRA |
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| Source name |
GM12878
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| Organism |
Homo sapiens |
| Characteristics |
cell line: GM12878
cell type: lymphoblast cell
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| Growth protocol |
GM12878 (HG001) cell line and GM24385 (HG002) cell line were cultured in RPMI-1640 (Gibco; 11875093) supplemented with 15%fetal bovine serum (FBS,Gibco,cat#26140079) and Penicillin/Streptomycin (PS; Gibco, cat#15140122). K562 cell line was cultured in RPMI-1640 supplemented with 10% FBS and 1% PS. COLO320DM cell line was cultured RPMI-1640 supplemented with 10% FBS and 1% PS. Wild-type V6.5 murine embryonic stem cells (mESCs) were cultured on 0.25% gelatin-coated 6-well plate in Dulbecco’s modified eagle’s medium (DMEM/F-12, Gibco, cat#11320033) containing 15%FBS,1%Glutamax(Gibco, cat#35050061),0.1mM 2-mercaptoethanol (Gibco, cat#21985023),1%MEM nonessential amino acids(Gibco, cat#11140050),1%nuleoside (Millipore, cat#ES-008-D), 1%PS and 10000× 107 units/ml Leukemia Inhibitory Factor (LIF; Millipore, cat#ESG1107). The other male mouse ES cell line (named F15) was cultured in serum-free 2i/LIF medium, with in equal volume of DMEM (DMEM/F-12, Gibco; 11320033) and Neurobasal Medium (Gibco; 131 21103049) maintained in 2i (3 μM CHIR99021 and 1 μM PD0325901), LIF (1 000 U/ml), as previously described. These cell lines were all cultured at 37 ˚C with 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Resuspended ligated nuclei pellets in 200μL 0.25%BSA in PBS each tube and added 0.2μL 1000×DAPI for sorting nuclei. Single nucleus was flow sorted into each well of 96-well plate containing 2 μL lysis buffer(0.04 μL 1M Tris-HCl (pH 8.0),0.08μL 500 mM NaCl,0.03μL10% Triton X-100 (Sigma), 0.0004μL 0.5 M EDTA,0.05μL 20mg/mL QIAGEN protease). And then to decrosslinking and digest, incubated at 50°C, 3h; 70°C, 20 min; 68°C, 45 min; 4°C pause. After digesting, 96-well plates were stored at −80 ˚C.
A single cell was flow sorted into each well of 96-well plate containing 2.5 μL lysis buffer (0.25 μL 100 mM Tris-EDTA, 0.075μL 10% Triton X-100, 0.05μL 1 M KCL, 0.125μL 20mg/mL QIAGEN protease). And then incubated at 50°C for 3h; inactivate the protease at 70℃ for 30 min; 4°C pause. After digestion, we added 15μl amplification mixture (1.5μL 10×Thermopol buffer, 0.6μL 2.5 mM dNTP, 0.2μL 100 mM MgSO4, 0.5μL 10 μM GAT-N5-3G primer, 0.5μL 10 μM GAT-N5-3T primer, 0.5μL 2 U/μl DeepVentR (exo-) polymerase; New England BioLabs, M0259L ) into each tube containing lysed single cell, and performed quasilinear pre-amplification: 95℃, 3 min; then 11 cycles of 4°C for the 50s, 10°C for 50s, 20°C for 50s, 30°C for 50s, 40°C for 45s, 50°C for 45s, 65°C for 4min, 95°C for 20s, 58°C for 20s. And then added 15μl similar amplification mixture within GAT-8nt-barcode-primer, the products were then exponentially amplified for 12 cycles of 95°C for the 20s, 58°C for 30s,72°C for 3min, and finally 72°C for 5 minutes. After amplification, we pooled products with different barcodes together and then purified them with DNA Clean & concentration-5 kit (Zymo Research, D4014) with 5× binding buffer. cDNA products were further used to construct of libraries for next-generation sequencing as the previous report
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
MALBAC
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| Data processing |
The raw sequencing data of MALBAC was preprocessed as previously described. Firstly, UMI-tools (v1.1.1) was used to extract the cell barcodes in read 2 and attached them to the read name of matched read 1, and a whitelist containing 96 cell barcodes were used to filter the reads. Next, fastp (v0.20.1) was used to perform quality control with default parameters and the output ‘clean’ fastq files were mapped to GRCh38 reference with ‘bwa mem’ (v 0.7.17-r1188), and only alignments with MAPQ > 30 were retained for downstream analysis. We also used picard MarkDuplicates (v2.23.6) to remove PCR duplicates. After that, we attached the cell barcodes in read name into the bam tag ‘CB’ with local python scripts (using the simplesam module) and split bam into single cells with bamtools (v2.5.1). The demultiplexed bam files of each cell were then used to calculate the copy number ratios by Control-FREEC (v11.6) in 1 Mb resolution with recommended parameters.
Assembly: GRh38
Supplementary files format and content: CNV ratio calculated by Control-freec at 1 Mb resolution for each cell
Library strategy: MALBAC
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| Submission date |
Jun 20, 2023 |
| Last update date |
Jul 14, 2023 |
| Contact name |
Jiansen Lu |
| E-mail(s) |
jiansenlu@pku.edu.cn
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| Organization name |
Peking University
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| Department |
Biomedical Pioneering Innovation Center (BIOPIC)
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| Lab |
Fuchou Tang
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| Street address |
No. 5 Yiheyuan Road, Haidian District
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| City |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE217189 |
scNanoHi-C: a single-cell long-read concatemer sequencing method to reveal high-order structures within individual cells |
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| Relations |
| BioSample |
SAMN35813892 |
| SRA |
SRX20733068 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7500657_GM12878_09-2_control_freec.tar.gz |
931.4 Kb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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